1. Field of the Invention
The invention relates to hyaluronic acid and more particularly to the microbiological transformation of organic precursors into hyaluronic acid.
2. Brief Description of the Prior Art
Hyaluronic acid is the main component of the capsules formed by many strains of group A and by most strains of group C Streotococci. These microorganisms can assimilate glucose and under a variety of environmental conditions produce hyaluronic acid as a secondary metabolite; see for example the description given by Roseman et al., J. Biol. Chem. 203, 213 (1953).
The synthesis of hyaluronic acid by Streptococci is influenced by many variable factors, genetic as well as nutritional. In spite of many reports concerning the optimal conditions for hyaluronic acid production by Streptoccocci, not all of the conditions have heretofore been defined.
In regard to genetic factors, it is well known that certain strains of Streptococci produce hyaluronic acid in one period of their life and secrete hyaluronidase at a later time; see for example: Pike, R. M., Hyaluronidase and Hyaluronic Acid of group A Streptococci., Am. J. Med., 4, 468. (1948). As one would expect, when extracellular hyaluronidase negative strains of Streptococci are grown under controlled conditions, yields of a high molecular weight hyaluronic acid are reportedly obtained; see U.S. Pat. No. 4,782,046 of Brown, et al.
The environmental factors necessary for the production of hyaluronic acid by Streptococci have been reported as including both anaerobic fermentation conditions and aerobic fermentation conditions. Under anaerobic conditions, hyaluronic acid yields of from 0.3 to 1.0 gms/liter of fermentation media have been reported; see for example Thonard et al., J. Biol. Chem., 239, 726 (1964); Holmstrom et al., Microbio., 15, 1409 (1967); and Kjems and Lebech, Acta Path. Microbiol. Scand., 84, 162 (1976). The hyaluronic acid obtained under these procedures generally has a weight average molecular weight of 700,000 or less [calculated from the limiting viscosity number; method of Laurent et al, Biochimica et Biophysica Acta, 42, 476 (1960)]. Under aerobic fermentation conditions, a product with a higher weight average molecular weight (circa. 2,000,000 or more) is reportedly obtained in comparable overall yields; see Japanese Patent publication Kokai No. 58-056692 (Apr. 4, 1983) filed by Akasaka et al. The higher molecular weight product is advantageous for a number of commercial purposes.
At the time of the Akasaka et al. publication, a debate arose as to the role of aeration in the culturing of the hyaluronic acid producing Streptococci. An earlier publication of Cleary et al., J. of Bacteriology, 140, No.3, pgs 1090-1097 (1979) had suggested that hyaluronic acid functioned as an oxygen impervious shield, protecting the Streptococcus from the toxic effect of molecular oxygen. Thus, as a cell protective mechanism it was thought that aerobic conditions stimulated hyaluronic acid production. There are artisans who subscribe to this theory, and data can be pointed to in support of the proposition. In fact our own work may be at least in-part supportive of the theory.
However, Bracke et al. (U.S. Pat. No. 4,517,295; May 14, 1985) discovered that improved yields of hyaluronic acid (reported as "a minimum" of 2 gms/liter of culture broth) were obtained under carbon dioxide enriched, anaerobic conditions. There is therefore controversy as to the role of aerobic and anaerobic conditions in the biosynthesis of hyaluronic acid employing the Streptococcus organism.
We have discovered that even higher yields of hyaluronic acid can be produced by Streptococcus by and through control of aerobic conditions during cultivation of the microorganism. Although we are not to be bound by any theory of operation, we believe the process of the invention and its advantageous yields may be due in part to the following explanation.
Streptococci, which are known pathogens, are preferably, cultivated in closed systems. The system may be initially charged with nutrients and sealed. A seed culture is introduced into the sealed system and cultivation encouraged. Metabolic products (other than carbon dioxide) are usually not removed from the system during cultivation.
In the aerobic cultivation of Streptococci in a closed system, charged with a total requirement of nutrient at the start of cultivation, there is a typical sequence of growth and metabolic events. Initially, a so-called "lag phase" occurs during which there is a linear growth of the microorganism at a relatively slow rate. The culture medium during this lag phase generally has a high dissolved oxygen content because there is a prevailing tendency to saturate the liquid nutrient medium with dissolved oxygen prior to initiating fermentation. In the lag phase there is usually a relatively high production of hyaluronic acid by the microorganism, in proportion to the biomass, i.e.; the weight of viable cells present. This may be due at least in part, to the high oxygen presence (Cleary et al. supra.).
At some point, usually after about 4 to 8 hours into the lag phase, there occurs exponential cell growth with an observable increase in the total biomass (termed the exponential phase). During the lag and exponential phases the microorganism is utilizing nutrient and oxygen for cell growth (division), maintenance and secondary metabolism. During the exponential phase hyaluronic acid is produced and secreted into the culture medium in substantially parallel proportion to the increase in biomass. This is in contrast to the higher proportional production of hyaluronic acid associated with the lag phase and could be expected since the cell activity is focused on use of nutrients for cell division and not secondary metabolism. The cell metabolism is primarily directed toward reproduction, in the exponential phase.
As nutrient is depleted from the growth medium, growth can no longer be sustained exponentially. The increase in cellular biomass peaks, levels off in a so-called "stationary phase" and then declines in a terminal or death phase as the microorganisms die faster than they reproduce. Nutrient is depleted to the point where only cell maintenance continues, for cell viability.
From all of the above, the skilled artisan might surmise that the best overall yields of hyaluronic acid by the Strectococcus would result if one were to cultivate the oxygen-sensitive microorganism under anaerobic conditions during the exponential phase to obtain an optimal exponential phase increase in biomass and then to provide aeration to provoke the higher population of the microorganism to maximize hyaluronic acid secretion. It was our discovery that a different sequence provides the better overall yields of the desired product, and that strict anaerobic conditions are not required or desired at all. Indeed, if one initiates the cultivation and the start of the exponential phase of growth in the presence of relatively high oxygen concentrations and then subsequently starves the growing biomass of oxygen during the exponential growth phase, higher overall yields of hyaluronic acid are obtained. The reduction of oxygen availability at a point of time during exponential growth actually raises the level of hyaluronic acid production, by the microorganism.